Method for producing optically active norborneol by ester hydrolysis

ABSTRACT

A method for producing an optically active norborneol is provided, which includes the step of bringing a microorganism or treated cells thereof into contact with (±)-exo-norbornane type ester represented by Formula (I), wherein the microorganism is selected from the group consisting of the genus Pseudomonas, the genus Acetobacter, the genus Arthrobacter, the genus Rhodotorula, and the genus Saccharomyces. According to this method, (+)- and/or (-)-exo-norbornane type alcohol can be obtained with high yield and high purity by a simple treatment.

TITLE OF INVENTION

A method for producing optically active norborneol this application is a371 of PCT/JP92/01588 filed Dec. 4, 1992.

TECHNICAL FIELD

The present invention relates to a method for producing optically activenorborneol by treating exonorbornane type ester with a microorganism.

BACKGROUND ART

Among methods for producing norborneol, the following chemical synthesismethod is known: Norbornane as starting material is reacted with anorganic acid to form an ester of norbornane; and then, this ester ischemically deacetylated to obtain norborneol. According to such achemical synthesis method, four kinds of stereoisomers ((+, -)-(endo,exo)-norborneol) are generated, so that complicated purification stepsare required for obtaining optically active norborneol by chemicalsynthesis.

As a method for biologically producing norborneol, the following methodis known: An ester of norbornane is brought into contact with amicroorganism or reacted with an enzyme and the ester is hydrolyzed. Inrecent years, Th. Oberhauser et. al. reported a method for producing(-)-norborneol from (+)-norbornyl acetate by using lipass derived fromCandida cylindraceae (Th. Oberhauser et. al., Tetrahedron, 43, 3931-39441987). Moreover, Oritani et. al. reported a method for producing(-)bornsol and (-)isoborneol from (±)bornyl acetate and (±)isobornylacetate by using a culture of Trichoderma sp, Trichoderma koningi,Bacillus subtilis var. Niger, and Absidia hyalospora (T. Oritani et.al., Agr. Biol. Chemo, 38(10), 1961-1964, 1974). However, the reactionshave low selectivity and thus obtained products have low optical purity.Japanese Laid-Open Patent Publication No. 2-273196 discloses an opticalresolution method in which an inhibitor, for selectively inhibiting thereaction of one of the enantiomers by a biological catalyst, resolves aracemic compound. This method can be used for preparing optically activenorborneol. However, this method has the following disadvantages:screening is required in order to obtain an inhibitor effective for theselection of optically active norborneol; and an inhibitor should beremoved in the course of purification after the reaction, thuscomplicated manipulation is required. In view of these circumstances,there has been a demand for an improved biological method for obtainingoptically active norborneol, in which an ester is brought into contactwith a microorganism or enzyme with high stereoselectivity in the courseof the hydrolysis of the ester.

DISCLOSURE OF INVENTION

According to the present invention, a method for producing opticallyactive norborneol with high purity by treating an exo-norbornane typeester with a microorganism is provided.

A method for producing an optically active norborneol of the presentinvention includes the step of bringing a microorganism or treated cellsthereof into contact with (±)-exo-norbornane type ester represented byFormula (I): ##STR1##

where R represents an acyl group, A and B represent independentlyhydrogen or A and B are taken together to form a chemical bond,

wherein the microorganism is selected from the group consisting of thegenus Pseudomonas, the genus Acetobacter, the genus Arthrobacter, thegenus Rhodotorula, and the genus Saccharomyces.

According to the present invention, (2S)-exo-norbornane type ester isselectively hydrolyzed by bringing (±)-exo-norbornane type estersrepresented by the above-mentioned Formula (I) into contact with theabove-mentioned microorganisms or treated cells thereof. Due to thishydrolysis, optically active alcohol and unchanged (2R)-exo-norbornanetype ester are obtained.

According to the above-mentioned method, exo-norbornane type alcohol or(2R)-exo-norbornane type ester is obtained from the reaction solution.

(±)-Exo-norbornane type ester used in the present invention isrepresented by Formula (I): ##STR2##

where R represents an acyl group, A and B represent independentlyhydrogen or A and B are taken together to form a chemical bond.

In Formula (I), the acyl group is an aliphatic acyl group with 2 to 10carbon atoms, and preferably an aliphatic acyl group with 2 to 7 carbonatoms. Examples of the aliphatic acyl group include acetyl, propionyl,butyryl, isobutyryl, pentanoyl and hexanoyl. More preferred examples ofthe aliphatic acyl group include formyl, acetyl, propinonyl andisobutyryl. The acyl group may be a cycloalkyl carbonyl group with 4 to10 carbon atoms, and more preferably a cycloalkyl carbonyl group with 4to 7 carbon atoms. Examples of the cycloalkyl carbonyl group includecyclopropane carbonyl, cyclobutane carbonyl, cyclopentane carbonyl, andcyclohexane carbonyl. Alternatively, aryl carbonyl group, and morepreferably an aryl carbonyl group with 7 to 11 carbon atoms can be used.Examples of the aryl carbonyl group include benzoyl, p-toluoyl andnaphthoyl.

The above-mentioned compound is commercially available or can easily beproduced by chemical synthesis. For. example, norbornene is reacted withan appropriate organic acid (e.g., formic acid, acetic acid, propionicacid and butyric acid) in the presence of an acid catalyst; or a vinylester of an appropriate organic acid and cyclopentanediene are subjectedto a Dieis-Alder reaction, whereby the desired (±)-exonorbornane typeester (I) can be obtained with high yield.

According to the present invention, a microorganism selected from thegroup consisting of the genus Pseudomonas, the genus Acetobacter, thegenus Arthrobacter, the genus Rhodotorula and the genus Saccharomyces isused. Pseudomonas aeruginosa, Acetobacter pasteurianus, Arthrobacters.p, Rhodotorula pallida, Rhodotorula rubra or Saccharomyces sp. arepreferably used. In particular, Acetobacter pasteurianus andArthrobacter sp. are more preferably used.

More specifically, strains such as Pseudomonas aeruginosa IFO 12582,Acetobacter pasteurianus ATCC 9432, Acetobacter pasteurianus ATCC 12873,Arthrobacter sp. SHS-0145 (FERM BP-4060), Rhodotorula pallida IFO 0715,Rhodotorula rubra IFO 1101, .Rhodotorula rubra ATCC 2510 andSaccharomyces sp. SHS-20030 (FERM BP-4061) are used. The Arthrobactersp. SHS-0145 and Saccharomyces sp. SHS-20030 were respectively depositedin the Ministry of International Trade and Industry (MITI), Agency ofIndustrial Science and Technology, Fermentation Research Institute,located at 1-3, Higashi/chome Tsukaba-shi, Ibaraki ken 305, Japan withthe accession No. FERM BP-4060 and FERM BP-4061 on Nov. 2, 1992, inaccordance with Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure.

The above-mentioned microorganisms are grown in a medium containingnutrients generally used for growing microorganisms, such as glucose,sucrose, blackstrap molasses, polypeptone, meat extract, yeast extract,pork meat powder, soybean powder, phosphate salts, magnesium, iron andthe like.

In a method of the present invention, (±)-exo-norbornane ester isbrought into contact with the above-mentioned microorganism or treatedcells thereof. More specifically, for example, (±)-exonorbornane typeester (I) is directly added to the culture of the above-mentionedmicroorganism so as to provide a concentration of 0.5 to 10% and theculture thus obtained is further cultured, whereby the ester ishydrolyzed to obtain norbornane type alcohol 2S-(II) and norbornane typeester 2R-(I). Instead of the above culture, a cell suspension or a cellextract can be used for this hydrolysis. The cell suspension is obtainedby collecting cells from the culture by centrifugation, filtration, etc.and suspending the collected cells in a buffer the pH of which is to besuitable for the reaction or a buffer containing an organic solvent. Thecell extract is prepared by breaking the collected cells by employinglyric enzyme or generally using ultrasonification, French press, etc.and removing cell debris by centrifugation, etc.

In the present invention, bringing (±)-exo-norbornane type ester intocontact with a microorganism refers to directly adding(±)-exo-norbornane type ester to a culture; and bringing(±)-exo-norbornane type ester into contact with treated cells of amicroorganism refers to adding (±)-exo-norbornane type ester to a cellsuspension or a cell extract instead of the culture.

In order to allow the reaction to be efficiently conducted, (±)-ester(I) to be added can be used by being dissolved in a hydrophobic solventsuch as n-hexane or a hydrophilic solvent such as dimethyl sulfoxide,methanol and ethanol so as to provide a concentration of 10 to 75%.

A reaction process of the above-mentioned hydrolysis according to thepresent invention is shown in the following reaction process diagram a:##STR3##

In this process diagram, R, A and B are the same as those describedbefore.

As shown in the above-mentioned process diagram, among exo-norbornaneester, only 2S-(I) is selectively hydrolyzed and converted toexo-norbornane type alcohol 2S-(II). Most of norbornane type ester2R-(I) remains unreacted in a reaction solution. Thus, the reactionsolution containing norbornane type alcohol P2-S- (II) and norbornanetype ester 2R-(I) is obtained.

Alcohol 2S-(II) thus obtained and exo-norbornane type ester 2R-(I) whichremains unreacted can be separated from each other by known methods. Forexample, alcohol 2S-(II) and exo-norbornane type ester 2R-(I) can beseparated by chromatography. In addition, it is also possible thatalcohol 2S-(II) is made water-soluble by an appropriate method (e.g.,alcohol 2S-(II) is reacted with an organic acid such as phthalicanhydride to form a half ester), and then alcohol 2S-(II) thus obtainedcan be extracted to obtain a desired optically active norbornane typecompound.

Hereinafter, the case where (±) -exonorbornyl acetate is used will bedescribed. ##STR4##

As shown in the reaction process b, 0.1 to 2 times by volume of anappropriate solvent is added to the reaction mixture obtained by thetreatment of the present invention to extract norborneol(-)-2 andnorbornyl acetate(-)-1. As the solvent, chloroform, dichloromethane,ethyl acetate, n-hexane, etc. can be used alone or in combination. Theextracted norborneol (-)-2 and norbornyl acetate(-)-1 are appropriatelyconcentrated. Then, 1 to 2 times by volume of an organic acid anhydridesuch as maleic anhydride and phthalic anhydride is added to theconcentrate thus generated to form a water-soluble adduct ofnorborneol(-)-2 and an organic acid (maleic acid, phthalic acid, etc.).Moreover, a solution of a basic compound such as sodium bicarbonate isadded to the reaction mixture to be neutralized. Then, the organic acidadduct of norborneol(-)-2 and norbornyl acetate(-)-1 shift to an aqueouslayer and an organic solvent layer, respectively. The aqueous layer isseparated and subjected to an alkali treatment. After this, the aqueouslayer thus treated is extracted with an appropriate organic solvent suchas dichloromethane and concentrated; as a result, norborneol(-)-2 withhigh optical purity can be obtained. Norbornyl acetate(-)-1 can becollected from the organic solvent layer, which is obtained by beingseparated from the aqueous layer, by concentrating the solvent layer.After the collection, norbornyl acetate(-)-1 is brought into contactwith commercially available lipase, esterase, etc. or chemicallydeacetylated, whereby norborneol (+)-2 with high optical purity caneasily be obtained.

When (-)-exo-norborneol or (+)-exo-norborneol thus obtained is purified,if desired, by known methods such as that described in Irwin A., JonesJ. B.: J. Am. Chem. Soc. 98, 8476-8481 (1976), etc., an optically activesubstance with higher purity can easily be obtained. For example, thesecompounds are converted into water-soluble compounds by theabove-mentioned method. Then, (-)- or (+)-phenetylamine is added to therespective compounds thus obtained to form respective phenetylaminesalts, whereby an optically active substance with high purity of 98%enantiomeric excess (ee) or more can be obtained. Alternatively, thesecompounds can be purified by generally using column chromatography.

Hereinafter, the present invention will be described in detail by way ofillustrating examples and reference examples. The present invention isnot limited thereto.

EXAMPLE 1

Acetobacter pasteurjanus ATCC 9432 was inoculated into 3 L of GPBYmedium (4% D-glucose, 2% polypeptone, 2% meat extract, 2% yeast extract,0.2% potassium phosphate, 0.2% magnesium sulfate, and 0.2% calciumcarbonate, pH 7.0) previously provided in a 5 L small jar fermentor andcultured at 28° C. for 24 hours. The culture was centrifuged at 1,000 Gfor 10 minutes to remove a solid content, and then, centrifuged at ahigh speed of 20,000 G for 15 minutes to collect cells. The collectedcells were washed twice in a saline. As a result, 19.8 g of wet cellswere obtained. The wet cells were suspended in 600 ml of phosphatebuffer (0.1 M, pH 6.5). To this suspension, 12.0 g of (±)-exo-norbornylacetate ((±)-1) was added and the mixture was allowed to react withstirring at 30° C. for one hour. In the course of the reaction, 0.1Nsodium hydroxide was added in small portions to the reaction mixture andthe pH of the reaction mixture was maintained at 6.5±0.2. After thecompletion of the reaction, 1 ml of the reaction mixture was taken. To 1ml of the reaction mixture, 1 ml of chloroform was added to extractreaction products of norborneol(-)-2 and norbornyl acetate(-)-1. Theextracts were analyzed by gas chromatography (Column for opticalresolution CDX-B type (o0.25 mm×30 m), column temperature of 50° C. to210° C., manufactured by J & W Corp.), revealing that 8.5 mg/ml ofexo-norbornyl acerarc, 5.2 mg/ml of (-)-(1S,2S,4R)-exo-norborneol(- )-2,and 0.3 mg/ml of (+)-(1R,2R,4S)-exo-norborneol(+)-2 were detected. Thereaction mixture was extracted with toluene twice (300 ml and 100 ml).The toluene layers obtained by these extractions were attached to eachother to obtain 380 ml of toluene solution. The toluene solution wasconcentrated to about 50 ml. Then, 4.2 g of maleic anhydride was addedto the concentrated solution and allowed to react at 115° C. for 4hours. After that, to this reaction solution, 300 ml of saturated sodiumbicarbonate solution was added, stirred for 5 minutes, and allowed tostand for 10 minutes. After the reaction, an aqueous layer was separatedfrom a toluene layer, whereby about 300 ml of an aqueous layer and about50 ml of a toluene layer were obtained.

To 200 ml of the aqueous layer thus obtained, conc. hydrochloric acidwas added, whereby the aqueous layer was made acidic. The acidic aqueouslayer was extracted with 100 ml dichloromethane and concentrated toobtain 5.9 g of maleic acid compound. The maleic acid compound thusobtained was dissolved in 50 ml of methanol. Then, 4.0 g of potassiumhydroxide dissolved in 50 ml of water was dropped to the solution ofmaleic acid compound, and allowed to react at room temperature for 30minutes. After the completion of the reaction, the reaction product wasextracted with dichloromethane and the extract was concentrated toobtain colorless crystal of (-)-(1S,2S,4R)-exo-norborneol(-)-2. Yield:2.8 g (64%), [α]²⁵ D: -3.01° (C=1.46, CHCl₃), and optical purity: 90%ee.

EXAMPLE 2

Norbornyl acetate and norborneol were extracted from 400 ml of thereaction mixture, which contains the cell suspension and(±)-exo-norbornyl acetate ((±)-1) obtained in the same way as in Example1, with 300 ml of toluene in the same way as in Example 1. The extractwas concentrated to about 50 ml. To the concentrated solution, 4.2 g ofphthalic anhydride was added and allowed to react at 115° C. for 4hours. After that, 200 ml of saturated sodium bicarbonate solution wasadded to the reaction solution, stirred at room temperature for about 5minutes, and allowed to stand for 10 minutes. After the reaction, atoluene layer was separated from an aqueous layer, whereby 50 ml of atoluene layer and 200 ml of an aqueous layer were obtained. To 200 ml ofthe aqueous layer thus obtained rained, conc. hydrochloric acid wasadded, whereby the aqueous layer was made acidic. After that, the acidicaqueous layer was extracted with 100 ml of dichloromethane and thedichloromethane was distilled off to obtain 7.3 g of phthalic acidmonoester. The phthalic acid monoester thus obtained was dissolved in 75ml of ethyl acetate. After chat, 3.4 g of (-)-phenetylamine was droppedto the solution of the phthalic acid monoester to obtain 9.0 g ofphenetylamine salt. The amine salt was recrystallized twice with 45 mlof ethanol. Then toluene and water were added to the crystal thusobtained, and 8.3 ml of 7.3% solution of hydrochloric acid was droppedto the crystal, and after that, the toluene layer was separated. Thetoluene layer was concentrated and dissolved in 20 ml of methanol. Then,17.4% solution of sodium hydroxide was dropped to the methanol in whichthe toluene layer was dissolved and allowed to react at 50° C. for 2hours. The reaction product was extracted with dichloromethane, and theextract was concentrated to obtain 1.3 g of colorless crystal of(-)-(1S,2S,4R)-exo-norborneol(-)-2. Yield: 50%, [α]²⁵ D: -3.20° (C=1.30,CHCl₃), and optical purity: 98%ee.

EXAMPLE 3

First, 50 ml of the toluene layer obtained in Example 1 was concentratedunder reduced pressure to obtain 4.9 g of oil-like norbornyl acetate(-)-1. This norbornyl acetate was dissolved in 50 ml of methanol. Then,50 ml of 8% sodium hydroxide was dropped to the solution and allowed toreact at 50° C. for 2 hours. After that, the reaction product wasextracted with dichloromethane. The extract thus obtained wasconcentrated under reduced pressure to be crystallized to obtain 3.1 gof colorless crystal of (+)-(1R,2R,4S)-exo-norborneol(+)-2. Yield: 71%,and optical purity: 95%ee or more.

EXMAPLE 4

First, 1L of the GPBY medium described in Example 1 was placed in a 2 Lmini-Jar fermentor, and inoculated with Acetobacter pasteurianus ATCC9432, followed by being cultured at 30° C. for 24 hours. Then, 10.0 g of(±)-exo-norbornyl acetate was added to the culture and the culture wasallowed to react for another 4 hours. The reaction product thus obtainedwas extracted with 400 ml of chloroform. The extract was subjected togas chromatography, whereby the content of a compound contained in theextract was measured. This revealed that 10.3 mg/ml of exo-norbornylacetate, 6.2 mg/ml of (-)-(1S,2S,4R)-exo-norborneol(-)-2, and 0.5 mg/mlof (+)-(1R,2R,4S)-exo-norborneol(+)-2 were obtained.

EXAMPLE 5

First, 20 ml of 0.1 M tris hydrochloride buffer (pH 7.2) was added to 10g of wet cells of Acetobacter pasteurianus ATCC 9432 obtained in thesame way as in Example 1 to prepare a cell suspension. The cells werebroken by a French press. Then, 0.3 mg of deoxyribonuclease was added toabout 30 ml of the lysate thus obtained. The lysate was treated on icefor 20 minutes and centrifuged at 30,000 G for 15 minutes, wherebybroken cells and cell debris were removed to obtain about 22 ml of cellextract. Then, 100 mg of (±)-exo-norbornyl acetate was added to 10 ml ofthe cell extract and allowed to react with gentle stirring in anincubator at 30° C. for one hour. After the completion of the reaction,the reaction product was extracted with 10 ml of chloroform. The extractwas subjected to gas chromatography, whereby the content of a compoundcontained in the extract was measured. This revealed that theconcentrations of exonorbornyl acetate,(-)-(1S,2S,4R)-exo-norborneol(-)-2, and(+)-(1R,2R,4S)-exo-norborneol(+)-2 were 4.6 mg/ml, 3.0 mg/ml, and 0.3mg/ml, respectively.

EXAMPLE 6

First, 50 ml of the GPBY medium described in Example 1 was placed in 500ml flask and then sterilized. The medium was inoculated with amicroorganism shown in Table 1 and cultured at 30° C. for 24 hours.Then, 500 mg of (±)-exo-norbornyl acetate was added to the culture andcultured for 16 hours after being sealed with a rubber stopper. Thenorbornyl acetate and norborneol of the culture were analyzed by gaschromatography in the same way as in Example 1. The results are shown inTable 1.

                  TABLE 1                                                         ______________________________________                                                    Concentrations after reaction (mg/ml)                             Name of microorganism                                                                       NAC*      (-)-2      (+)-2                                      ______________________________________                                        Pseudomonas aeruginosa                                                                      6.80      1.60       0.28                                       IFO 12582                                                                     Acetobacter pasteurianus                                                                    6.20      2.24       0.12                                       ATCC 9432                                                                     Acetobacter pasteurianus                                                                    5.82      2.52       0.22                                       ATCC 12873                                                                    Rhodotorula pallida IFO                                                                     6.54      1.47       0.61                                       0715                                                                          Rhodotorula rubra                                                                           7.62      1.01       0.43                                       ATCC 2510                                                                     Saccharomyces sp.                                                                           7.88      1.04       0.28                                       SHS-20030 (FERM                                                               BP-4061)                                                                      ______________________________________                                         *NAC: Exonorbornyl acetate                                               

EXAMPLE 7

First, 1L of the GPBY medium described in Example 1 was placed in 2 Lmini-jar fermentor. Then, Pseudomonas aeruginosa IFO 12582 andSaccharomyces sp. SHS-20030 (FERM BP-4061) were respectively broughtinto contact with the medium and cultured at 30° C. for 24 hours. Eachculture was centrifuged as described in Example 1, whereby cells in eachculture were collected and washed. The cells from each culture weresuspended in a 0.05 M phosphate buffer (pH 7.0) so that the opticaldensity (660 nm) thereof be 10. (±)-exonorbornyl acetate was previouslydissolved in dimethyl sulfoxide so as to give a concentration of 50w/w%.

Next 100 ml of each cell suspension was placed in 500 ml flask, and 2.0ml of 50% (±)-exonorbornyl acetate solution was added to each cellsuspension. Each flask was sealed with a rubber stopper, and then, eachcell suspension was allowed to react at 30° C. The progress of thereaction was monitored based on the added amount of 0.1N sodiumhydroxide required for maintaining pH 7.0. At the end of the reaction, asmall amount of the reaction mixture (0.5 ml) was taken, and a reactionproduct was confirmed by gas chromatography in the same way as inExample 1. At a time when about 70% of added norbornyl acetate wasconsumed, the reaction was terminated and 100 ml of toluene was added tothe reaction mixture. Thus, norbornyl acetate and norborneol wereextracted. The extract from each reaction solution was concentrated toabout 25 ml. Then, 0.4 g of maleic anhydride was added to each extractand allowed to react at 115° C. for 4 hours. To each reaction solution,25 ml of saturated sodium bicarbonate was added and stirred at roomtemperature for 5 minutes, followed by being allowed to stand for about10minutes. As a result, an aqueous layer and a toluene layer were formedin each reaction solution. When the toluene layer was concentrated,oil-like (-)-exo-norbornyl acetate(-)-1 was obtained. This norbornylacetate was dissolved in 10 ml of methanol, and 8%.sodium hydroxide wasdropped to the solution thus obtained and allowed to react at 50° C. for2 hours in alkaline condition. Then, this reaction product was extractedwith 10 ml of dichloromethane. The extract was concentrated whereby(+)-(1R,2R,4S)-exo-norborneol(+)-2 was crystalized. The crystal thusobtained was analyzed by gas chromatography. The results are shown inTable 2.

                                      TABLE 2                                     __________________________________________________________________________                               (-)-2 crystal                                                     Concentrations                                                                            obtained by                                                       after reaction (mg/ml)                                                                    hydrolysis of NAC                                  Name of microorganism                                                                        NAC (-)-2                                                                             (+)-2                                                                             Optical purity                                                                       Yield*                                      __________________________________________________________________________    P. aeruginosa IFO 12582                                                                      3.22                                                                              2.98                                                                              1.10                                                                              98.0% ee                                                                             47%                                         Saccharomyces sp.                                                                            3.01                                                                              2.88                                                                              1.42                                                                              98.2% ee                                                                             38%                                         SHS-20030(FERM BP-4061)                                                       __________________________________________________________________________     ##STR5##                                                                 

EXAMPLE 8

First, 50 ml of Antibiotic medium 3 (Difco) was placed in a 500 mlflask. The medium was inoculated with Arthrobacter sp. SHS-0145 (FERMBP-4060) and cultured at 30° C. for 20 hours. After that, 100 mg of(±)-exo-norbornyl acetate was added to the culture, and the culture wasallowed to react for another 8 hours. To this culture was added 20 ml ofchloroform, whereby norbornyl acetate and norborneol were extracted. Therespective contents of (-)-(1S,2S,4R)-exo-norborneol(-)-2,(+)-(1R,2R,4S)-exo-norbo in the extract were measured by gaschromatography in the same way as in Example 1. This revealed that 1.02mg/ml of (-)-(1S,2S,4R)-exo-norborneol(-)-2 and 0.11 mg/ml of(+)-(1R,2R,4S)-exo-norborneol(+)-2 were contained in the extract.

Hereinafter, bacteriological characteristics of Arthrobacter sp.SHS-0145 (FERM BP-4060) will be described.

1. Morphological characteristics

Cells of this strain are Gram-positive rods and have various shapes andsizes. In an early logarithmic growth phase (bouillon agar medium, 30°C., 8-12 hours cultivation), the cells become rod-shaped with a size of1.0-1.1×3.0-4.0 μm. In a large logarithmic growth phase, the cellsbecome short rod (bouillon agar medium, 30° C., 24-48 hourscultivation). In a stationary phase, the cells become coccal (1.0-1.1 μmin diameter, bouillon agar medium, 30° C., 72-96 hours cultivation).Sporulation is not observed. The cells are motile.

2. Cultural properties

Growth on a bouillon agar plate (30° C., 7 days cultivation): Flatlustered circular colonies of light yellow to light yellowish white witha diameter of 1.4-1.6 mm are formed within 24 hours.

Growth on a bouillon agar slant (30° C., 7 days cultivation): The strainis moderately grown along an inoculation streak.

Growth in a bouillon soft agar (30° C., 7 days cultivation): The strainmoderately grows on a surface, and the growth is observed only on theupper portion of a stab culture.

Bouillon static cultivation (30° C., 7 days cultivation): The strainmainly grows to be a thick pellicle on a surface of the culture. Thebroth becomes moderately turbid, and the amount of a precipitate issmall. Odor is not particularly recognized.

3. Growth

Growth temperature: Possible growth temperature 10°-37° C., and optimumgrowth temperature 24°-32° C. (peptone water).

Heat resistance: The strain does not survive more than 30 minutes inskim milk at 63° C.

Growth pH: Possible growth pH 5.2-10, and maximum growth pH6.0-9.[(peptone water).

Behavior with respect to oxygen: aerobic. Growth in an anaerobicatmosphere (Gas pack®, bouillon agar plate) is not observed.

4. Composition of cell wall: meso-DAP (meso-diaminopimeric acid) is notdetected.

5. G+C content of DNA: 57%

6. Main various physiological characteristics of test: Oxidative(D-glucose)

Formation of acid: Acid is produced from D-glucose, galactose,D-mannose, sucrose, trehalose, fructose (weak) and mannitol (weak) butacid is hardly produced from L-arabinose and D-xylose. Acid is notproduced from lactose, ribose, sorbose, rhamnose, inositol, cellobiose,and maltose.

Litmus milk: Negative

Liquidization of gelatin: Negative

VP reaction: Negative

MR reaction: Negative

Production of hydrogen sulfide: Negative

Utilization of citric acid: Positive (Christensen medium, Simmonsmedium)

Utilization of inorganic N source: Ammonium salt and nitrate areutilized.

Urease: Positive

Catalase: Positive

Oxidase: Positive

DNase: Negative

Reduction of methylene blue: Positive

7. Source of Isolation: soil in Japan

Hereinafter, microbiological characteristics of Saccharomyces sp.SHS-20030 (FERM BP-4061) used in

Examples 6 and 7 will be described.

1. Morphological characteristics

Shape: This strain is a yeast in a spherical or oval shape of3.5-4.5×5-6 μm and mainly grows by budding. Multipolar budding isobserved (malt extract agar medium, 28° C., 3 days cultivation).Mycelium is not observed.

Ascospore: One to four spores in a spherical shape or nearly oval shapeare formed (Gorodokawa agar medium, 28° C., 10 days cultivation).

2. Assimilation and fermentation of sugar

The assimilation and fermentation of cose, galactose, sucrose, maltoseand raffinose are recognized. The assimilation and fermentation oflactose, cellobiose, and starch are not recognized.

3. Other Physiological test

Assimilation of paraffin: Negative

Assimilation of Potassium nitrate: Negative

Breakdown of arbutin: Negative

Demand for vitamin: None

INDUSTRIAL APPLICABILITY

The method for using the optically active norbornane type alcoholobtained according to the present invention will be described in thefollowing reference examples.

Reference Example 1

First, 2.8 g (0.025 moles) of (-)-(1S, 2S, 4R)-exo-norborneol obtainedin Example 1 was dissolved in 56 ml of methylene chloride. To thismixture, 8.1 g of pyridinium chlorochromate (PPC) (1.5 molar ratio) and1 g of molecular sieve 4A® were added and allowed to react at atemperature of 25 to 30° C. for one hour. The reaction solution wasdiluted with 56 ml of toluene, after which the reaction solution waspassed through a column of 28 g silica gel, whereby a substance whichwas not dissolved was removed. When the effluent solution wasconcentrated to be dried, crude (+)-norcamphor was obtained as whitecrystalline powder. The (+)-norcamphor thus obtained was dissolved in 45ml solution of tetrahydrofuran. The solution thus obtained was droppedto 30 ml solution of tetrahydrofuran of LDA (1.1 molar ratio) at -10° to-15° C. The solution thus obtained was allowed to react for 20 minutesat -10° to -15° C. After that, 3.3 g of allyl bromide (1.1 molar ratio)was dropped to the reaction solution at 0° C. or less and allowed toreact at room temperature for 3 hours. The reaction solution was flowninto 70 ml of ice water and was made acidic with dilhydrochloric acidsolution. After that, the reaction solution was twice extracted with 50ml of toluene. The toluene layer was washed with water and the solventwas distilled off under reduced pressure. As a result, crude(+)-exo-3-(2-propenyl)bicyclo[2,2,1 ]heptane-2-on was obtained as anoily residue. The (+)-exo-3-(2-propenyl)-bicyclo[2,2,1]heptane-2-on waspurified by collecting fractions at a boiling temperature in the rangeof 92° to 103° C./ to 12 mmHg under reduced pressure. Yield: 3.07 g(82%), chemical purity (GC): 98.6%, Endo isomer: 0.4%, optical purity(HPLC): 90%ee, and specific rotation: [α]²⁵ D+84.6° (C=1.0, CHCl₃).

Reference Example 2 .

The reaction and treatment were conducted by using(-)-(1S,2S,4R)-exo-norborneol obtained in Example 2 in the same way asin Reference Example 1. The crude product was purified by distillationunder reduced pressure, and fractions were collected at a boiling pointin the range of 74° to 86° C. /3 to 4 mmHg to obtain desired(±)-exo-3-(2-propenyl)-bicyclo[2,2,1]heptane-2-on. Yield: 1.38 g (78%),chemical purity (GC): 98.8%, Endo isomer: 0.9%, optical purity (HPLC):98%ee, specific rotation: [a]²⁵ D+89° (C=1.286, CHCl3), IR(Film) 3060,1740, 1640, 1460, 1440, 1310, 1090 cm⁻¹. ¹ H NMR(CDCl₃) δ1.30-2.00(m,8H), 2.5-2.6(m, 3H) δ4.90-5.20(m, 2H), 5.7-5.9(m, 1H).

The compounds obtained in the above-mentioned reference examples can beused by the method (J. Med. Chem. 31(9), 1847-1854(1988)) as TXA₂receptorantagonist which is pharmaceutically useful.

We claim:
 1. A method for producing a (2S)-exo-norborneol or(2R)-exo-norbornane type ester comprising the steps of bringing amicroorganism or cell extract or cell suspension thereof into contactwith a (±)-exo-norbornane type ester of the Formula (I): ##STR6## whereR is an acyl group, A and B are independently hydrogen or A and B form achemical bond,wherein the microorganism is selected from the group:consisting of Pseudomonas aeruginosa, Acetobacter pasteurianus,Arthrobacter sp. SHS-0145, Rhodotorula pallida, Rhodotorura rubra, andSaccharomyce sp. SHS-20030, and recovering the (2S)-exo-norborneol or(2R)-exonorbornane type ester.
 2. The method according to claim 1,wherein R is an acetyl group.
 3. The method according to claim 1 or 2wherein the microorganism is Acetobacter pasteurianus.
 4. The methodaccording to claims 1 or 2, wherein the microorganism is Arthrobactersp. SHS-0145 (FERM No. BP4060).
 5. A biologically pure culture ofArthrobacter sp. SHS-0145 (FERM No. BP4060).